Many RNA viruses usurp the host's protein synthesis (translation) machinery by bypassing the cellular translational control systems. The 5' cap and poly(A) tail on cellular mRNAs interact with translation factors to form a closed-loop mRNA structure in a regulated process necessary to recruit the ribosome and initiate translation. Many viral RNAs avoid this control step by lacking a 5' cap or poly(A) tail and instead harboring sequences in the untranslated regions (UTRs) that control translation by non-canonical means. Understanding how viruses do this could lead to development of antiviral agents specific to the unique viral translation mechanisms. This proposal focuses on the novel cap-independent translation element (TE) in the 3' UTR of barley yellow dwarf virus (BYDV) RNA that facilitates translation initiation at the 5' end. This research aims to determine how the TE (1) recruits translational machinery, and (2) communicates with the 5' end where initiation ensues. BYDV RNA forms the closed-loop structure by a novel means: direct base pairing between the 3' TE and the 5' UTR. This base pairing is necessary but insufficient to mediate translation in vivo. The first aim is to determine the tolerance of this closed-loop base pairing for sequence changes, and to distinguish the BYDV RNA sequences and structures that mimic 5' cap and poly(A) tail functions in vivo. The second aim is to identify the proteins that bind the TE, map their binding sites, and determine their roles. Preliminary evidence indicates that the TE may recruit the ribosome via factors that normally bind only to the 5' cap. The model to be tested is that factors and the ribosome are recruited to the 3' UTR and delivered to the 5' end by base pairing. The research will employ (and improve upon) established in vitro and in vivo translation assays, RNA replication assays, RNA structural analysis, RNA-protein binding and ribosome binding assays, proteomics, and structure-guided mutagenesis. This research on a model virus may contribute to means of controlling many human pathogens, including poliovirus and hepatitis C virus, that also employ cap-independent translation regulated by interactions between the UTRs. It also applies to nidoviruses and flaviviruses (e.g. Dengue, West Nile) that regulate gene expression and replication by long-distance RNA base pairing between UTRs. Finally, the research will provide fundamental insight on eukaryotic translation mechanisms.